Factors other than erythropoietin (Ep) have been shown to influence the early stages of erythropoiesis. Since bone marrow is the primary site of hemopoiesis, we assayed conditioned media obtained from human bone marrow suspension cultures and found them to contain factors which enhance the growth of early erythroid progenitors in culture. This is a convenient source of material because it contains undetectable amounts of Ep and granulocytic colony stimulating activity. We have purified the burst-promoting activity (BPA) from bone marrow conditioned media (BMCM) 500-1000 fold by ion-exchange chromatography on diethylaminoethyl (DEAE) Sephadex and adsorption chromatography on calcium phosphate gel. This partially-purified factor increases burst number and hemoglobin (Hb) synthesis at limiting serum concentrations. We now propose to continue the purification of marrow BPA in order to biochemically characterize the active component. An alternative source of BPA, available in larger amounts, is rabbit BMCM which we have found to be as active on human cells as human BMCM. We simultaneously plan to attempt to isolate larger amounts of BPA from crude urinary extracts prepared from the urine of aplastic anemia patients. Dr. M. Kawakita is currently preparing a crude protein fraction from 500 liters of urine obtained from aplastic anemia patients. From this material, we will attempt to purify a substantial amount of BPA in order to carry out a detailed biological characterization of its action. A preliminary study indicates that BPA is present in this material and that it can be separated from Ep by adsorption to calcium phosphate gel. Methods that have been useful in the purification of marrow BPA and urinary Ep will be used to purify urinary BPA. Once it is purified to homogeneity, we will initiate studies of its biological properties on cultured cells. For example, we plan to study the target of BPA action. Using preparations of high-specific activity, we will examine the relative sensitivity of erythroid precursors to differing maturational stages. Since we have reported that BMCM increases both total Hb synthesis and the relative rate of fetal Hb synthesis in culture, we also plan to study how pure BPA affects Hb synthesis.